Pepsinized collagen implants and biomedical uses thereof

ABSTRACT

Disclosed are pepsinized collagen implants, such as membranes and sponges, and methods of making them, which entails solubilizing pepsinized collagen in a buffer containing a polyol, e.g., mannitol or sorbitol, wherein the buffer has a substantially neutral pH. Methods of using the pepsinized collagen implants for clinical applications are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 14/209,320 filed Mar. 13, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/789,997, filed Mar. 15, 2013, thedisclosures of which are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Collagen is the most abundant protein in the extracellular matrix ofhuman tissue and plays important roles in providing structural supportas well as performing other functions in tissue growth and regeneration.Apart from collagen, other types of extracellular matrix components suchas proteoglycans, elastin, etc. also play important roles in maintainingtissue structure and function. Producing scaffolds simulating naturaltissue is an essential enabling technology in the tissue engineeringindustry.

Collagen is an excellent natural biomaterial for tissue engineeringbecause of its close resemblance to nature, low immunogenicity andexcellent biocompatibility. However, unprocessed collagen usually hasinsufficient mechanical properties for it to be useful in engineeringtissues in particular the weight-bearing tissues such as tendons,ligaments, intervertebral discs, etc. Unprocessed collagen is alsodifficult to manipulate and put sutures through during the implantationprocess. Further, unprocessed collagen is highly water swellable and isvulnerable to enzymatic digestion and thermal denaturation.

Known methods of making collagen membranes often involve extensiveextraction procedures and lengthy isolation steps to obtain specificforms of collagen. These procedures often involve coacervation ofcollagen fibers, de-fatting, multi-cycle vitrification, extensiveultracentrifugation, and electrochemical plating, which are timeconsuming, difficult to scale up, and expensive. Tanaka, et al.,Biomaterials 32:3358-66 (2011) reports on the production of transparentcollagen laminates prepared by oriented flow casting, multi-cyclicvitrification, and chemical cross-linking of atelocollagen, which asknown in the art is pepsin-solubilized Type I collagen. In addition tothe aforementioned disadvantages associated with multi-cyclicvitrification, proteolytic digestion of collagen with enzymes such aspepsin requires strongly acidic conditions. Aside from the insolubilityof pepsinized collagen in water, the acidic conditions presentdifficulties from the standpoint of preparing collagen implants thatcontain other bioactive materials.

Accordingly, there is a need in the art for methods of preparingcollagen implants that do not suffer from one or more of theaforementioned disadvantages.

BRIEF SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method ofpreparing a collagen implant for use in tissue repair, that entails (a)digesting collagen with pepsin, thus producing pepsinized collagen; (b)solubilizing the pepsinized collagen in a buffer composition having asubstantially neutral pH, and which comprises a polyol; (c) drying thethus-solubilized, pepsinized collagen, forming the pepsinized collagenimplant; and optionally (d) reacting the implant with a cross-linkingagent. The implants may be produced in the forms of membranes andsponges.

A second aspect of the present invention is directed to a collagenimplant made by the inventive process. The membranes may be formulatedto be transparent or semi-transparent, and shaped and sized for purposesof specific clinical applications, e.g., implants in the form of graftsor prosthetics. In some embodiments, the implant may be imparted withanti-adhesive properties. In these embodiments, the pepsinized collagenis formulated in the buffer composition that further includes ananti-adhesive agent, e.g., a sugar, a PEGylated amine, a PEGylatedcarboxylic acid, a short chain aliphatic acid anhydride or fatty acidanhydride, or a positively charged amino acid, or a copolymer orhomopolymer thereof.

Yet other aspects of the present invention are directed to methods ofusing the collagen implants. The pepsinized collagen sponges can be usedin clinical applications such as hemostasis and wound repair. Thepepsinized collagen membranes can be used in clinical applications suchas tissue repair or generation, or for promoting or facilitating tissuegrafts, that entail covering an area of damaged, injured, diseased,wounded, removed, or missing tissue of a body of a subject.

The methods of the present invention are advantageous in severalrespects, particularly with respect to implants based on Type Icollagen. Type I collagen exists in fibril form that is not soluble inaqueous media at pH 5 to 7.4. It is soluble in acidic media only at verylow concentrations. Applicant has surprisingly and unexpectedlydiscovered that the inclusion of a polyol in a buffer solution having asubstantially neutral pH solubilizes the pepsinized collagen readilyquickly and substantially completely, such that it undergoesself-assembly. Upon drying, the solution forms membranes or sponges.

There are several Type I collagen-based membranes which are not idealfor repair as they often result in capsule formation and ormineralization as they are fully developed collagen fibrils. Inaddition, they have been known to be immunogenic. The pepsinizedcollagen membranes of the present invention are relatively advantageousin these respects. The pepsinized collagen undergoes reassemblyinternally following implantation in the body so that the immunogenicityis far less compared to the stand alone Type I-based collagen products.Moreover, integration of the present membranes with the host tissue isexpected to be improved. Toughness and elasticity of these products mayalso be easily varied to accommodate a variety of clinical uses.

Even further, the pH range of the buffer does not adversely affect ordetract from the beneficial medicinal properties and uses of thecollagen implant. It also facilitates addition of other biomaterialsinto the buffer, for purposes of incorporating them into the collagenimplant.

In some embodiments, the collagen implants are transparent orsemi-transparent, which provides an advantage of allowing for the directand non-invasive monitoring of the healing process such as a woundvisually through the membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an embodiment of the method of thepresent invention.

FIG. 2 is a schematic illustration of an embodiment of the method of thepresent invention wherein the collagen implant has pendant glucuronicacid and glucosamine residues.

DETAILED DESCRIPTION

Collagen for use in the present invention may be obtained from a varietyof sources including, for example, bovine hide. Pepsinization ofcollagen may be performed in accordance with standard procedures. See,e.g., McPherson, et al., Collagen Rel. Res. 5:119-135 (1985) (describingpepsin solubilization of collagen using bovine corium). In someembodiments, bovine hides such as male bovine hides derived from acontrolled herd may be cold shipped with a maximum time allowed betweenslaughter and trimming, and then soaked in acetic acid in order tominimize degradation and to reduce bioburden. The soaked and trimmedhides may then be ground up and digested in a temperature andpH-controlled vessel using a pepsin citrate solution as the primarydigestive agent. A settling step may then be conducted, usingdiatomaceous earth to filter out extraneous cellular material, followedby a viral inactivation step e.g., using NaOH, to inactivate prions. Thepepsinized collagen may be further purified using known techniques,e.g., ultrafiltration at a pH of about 2.0.

The amount of pepsinized collagen that is added to the buffer generallyranges from about 0.1 mg to about 60 mg, and in some embodiments fromabout 1 to about 30 mg, and in yet other embodiments from about 2 toabout 20 mg, per 1 ml of buffer.

Buffers that may be useful in the present invention are known in theart. Representative examples of buffers include physiologicallyacceptable buffers such as saline, phosphate buffered saline, etc., andwhich may include salts such as sodium acetate, calcium acetate, sodiumsuccinate dibasic, and calcium propionate. The buffer is chosen orformulated so as to have a substantially neutral pH, which for purposesof the present invention, refers to a pH at which the pepsinizedcollagen is readily solubilized (e.g., in a matter of minutes at roomtemperature). The pH generally ranges from about 5 to about 8, and insome embodiments from about 5.5 to about 7.5, and in yet otherembodiments from about 6 to about 7.5, and in yet other embodiments fromabout 6 to about 7. The amount of buffer varies, depending on factorssuch as the amount of pepsinized collagen. Agents for adjusting the pH,e.g., HCl, may be used to achieve the desired pH. Other inert excipientsknown to those in the art may also be added.

The buffer solution contains a polyol. Useful polyols in the presentinvention include C₄-C₆ polyols, representative examples of whichinclude sorbitol, mannitol, and xylitol. The amount of the polyolgenerally ranges from about 1 to about 30 grams per 100 ml of buffer,and in some embodiments from about 2.5 to about 20 grams per 100 ml ofbuffer, and in yet other embodiments from about 5 to about 10 grams per100 ml of buffer (i.e., from about 5% to about 10% (w/v)).

A variety of other ingredients may be added to the buffer prior to thedrying step in order to be incorporated into the collagen implant. Suchingredients may be added to optimize one or more physical properties ofthe collagen membrane, as well as to provide certain medicinal ortherapeutic benefits depending upon the ultimate clinical application.In some embodiments, other types of collagen may be added such asnon-pepsinized collagen fibers, Type I collagen, Type II collagen, andType III collagen and Type IV collagen. For example, depending on thedesired physical properties (e.g., sponginess, elasticity, toughness),Type I collagen may be present. Type I collagens include native fibrousinsoluble human, bovine, porcine, or synthetic collagen, solublecollagen, reconstituted collagen, and microfibrillar forms of collagenas described, for example, in U.S. Pat. Nos. 6,096,309 and 6,280,727.The weight ratio of the Type I collagen to the pepsinized collagengenerally varies from about 0.01 to about 0.99, and in some embodimentsfrom about 0.1 to about 0.9, and yet in other embodiments from about 0.2to about 0.5.

Yet other types of additional ingredients, such as therapeuticallyactive or beneficial agents may be added and become incorporated intothe pepsinized collagen implants. Representative examples of such agentsinclude antibiotics, analgesics, anti-inflammatory agents, cells, growthfactors and other non-cellular agents. Active agents may be added to thebuffer composition before drying; they may be added after drying andbefore packaging; or (e.g., in the case of sponges) they may be addedsuitably prior to the clinical application or procedure.

Representative examples of antibiotics include tetracyclines such astetracycline, doxycycline and aureomycin; penicillins includingpenicillin V, ampicillin, amoxicillin, bacampicillin, cabenicillin,carbenicillin, cloxacillin, dicloxacillin, nafcillin and oxacillin;cephalosprins such as cephalexin, cephradine, cefadroxil, cefaclor,cefuroxime axetil, cepfodoxime, loracarbef and cefixime; aminoglycosidessuch as gentamycin sulfate, tobramycin, amikacin, netimicin andneomycin; polymicins such as polymixin B; and sulfonamides such asmafenide, silver sulfadiazine and sulfasalazine.

Representative examples of analgesics include morphine, narcoticantagonists (e.g., naloxone), local anesthetics (e.g., lidocaine,bupivacaine, mepivacaine, dibucaine, prilocaine, etidocaine,ropivacaine, procaine, tetracaine, etc.), glutamate receptorantagonists, adrenoreceptor agonists, adenosine, canabinoids,cholinergic and GABA receptors agonists, and neuropeptides.

Representative examples of anti-inflammatory agents includeanti-cytokine agents such as TNF-α inhibitors, IL-1 inhibitors, IL-6inhibitors, IL-8 inhibitors, IL-12 inhibitors, IL-15 inhibitors, IL-10,NF-κβ inhibitors, and interferon-gamma (IFN-gamma).

The compositions may further include cells. Cells may be autologous,allogenic, xenogenic, or a combination thereof. Suitable types of cellsinclude, for example, stem cells, mesenchymal stem cells, and/orprogenitor cells, including cells derived from bone marrow, synovialfluid, synovium, placenta, umbilical cord, skin, muscle, and fat/adiposetissue, Schwann cells, endothelial cells, epithelial cells, Sertoli'scells, fibroblasts, or any cells useful or desirable in applications fortissue repair or regeneration. Cells may also be differentiated cellsincluding, for example, chondrocytes, tenocytes, osteoblasts, andsynoviocytes. The composition may include one type of cells or acombination of two or more cell types.

The compositions may further include other, non-cellular therapeuticallybeneficial agents such as growth factors (e.g., TGF-β, EGF, FGF, IGF-1BMP-7 and OP-1, etc.), glycosaminoglycans (GAGs) (e.g., aggrecan,decorin, biglycan and fibromodulin), chemokines and cytokines (e.g.,interleukins and interferons), chitosan and hyaluronan. Extracellularmatrix molecules that bind to growth factors, e.g., heparan sulfate andproteoglycans, may advantageously be added to serve as a reservoir forthe factors.

In some embodiments, the collagen implant may be prepared so as topossess anti-adhesive properties, which in the context of the presentinvention, refers to substantially reduced adhesion to neighboringtissues and organs. To impart the anti-adhesive property, the buffercomposition may be formulated so as to further include an anti-adhesiveagent. Examples of such agents include saccharides, e.g.,monosaccharides such as mannose, fucose, glucose, galactose, glucuronicacid, glucosamine, and N-acetyl glucosamine. Glucuronic acid andN-acetyl glucosamine are the monomeric building blocks of hyaluronicacid. The saccharide may be added to the buffer in the form of aphysiologically acceptable salt, e.g., a hydrochloride salt. Otherexamples of anti-adhesive agents that may be suitable for use in thepresent invention include anhydrides of short-chain aliphaticdicarboxylic acids such as maleic acid, succinic acid, and itaconicacid, as well as fatty acid anhydrides (e.g., anhydrides of saturated orunsaturated C₁₂-C₁₈ fatty acids).

Further examples of anti-adhesive agents that may be suitable for use inthe present invention include positively charged amino acids, e.g.,lysine, arginine, histidine and glutamic acid, and copolymers andhomopolymers thereof, e.g., polyarginine, polylysine, polyglutamic acid,and polyhistidine.

Further examples of anti-adhesive agents that may be suitable for use inthe present invention include polyethylene glycols derivatized withamine or carboxylic acid groups. Representative examples of PEGylatedamines may be represented by the formula

wherein n is an integer from 4 to 24, e.g., 4, 8, 12, or 24. Otherexamples of PEGylated amines may be represented by the formula:

wherein n is such that the average molecular weight (Mn) of thePEGylated carboxylic acid is about 1500.

Representative examples of PEGylated carboxylic acids that may besuitable for use in the present invention may be represented by theformula:

wherein n is such that the average molecular weight (Mn) of thePEGylated carboxylic acid is about 600. Other examples of PEGylatedcarboxylic acids may be represented by the formula below:HOOC—(CH₂CH₂O)_(n)—CH₂CH₂—COOHwherein n is an integer of from 4 to 24, e.g., 4, 8, 12 or 24.

The amount of the anti-adhesive agent present in the buffer compositionmay vary, e.g., depending on the desired degree of transparency. Ingeneral, the amount of this agent present in the buffer composition isfrom about 0.1 mg/ml to about 200 mg/ml, and in some embodiments, fromabout 0.5 mg/ml to about 50 mg/ml (w/v).

The degree of transparency may be achieved by adjusting the thickness ofthe membrane (which is influenced by the amount of the concentration ofthe pepsinized collagen in the buffer), the pH of the buffer, and thepresence of various additives such as the polyol and the salt(s) in thebuffer. The term “transparent” means that the composition issufficiently clear in the visible light range as to result in anabsorbance of less than 0.6 OD units when measured with light having awavelength at 410 nm through a 1 millimeter sample of the composition.

Following addition of the desired materials, the solution, which due tothe presence of the pepsinized collagen, is viscous in nature, is dried.Drying may be conducted in accordance with standard techniques such asair drying (at normal pressure or under vacuum) and lyophilization. Insome embodiments, sponges may be obtained through lyophilization. Insome embodiments, transparent and semi-transparent membranes may beobtained by air drying. The amount of time for drying will vary, e.g.,depending upon whether the solution is subjected to heat.

Following drying, the resultant collagen implant may be subjected tocross-linking for purposes of structural integrity. This step may beconducted in accordance with standard procedures and reagents in theart, e.g., 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC), glutaraldehyde (in liquid or vapor form), and genipin. The amountof cross-linking agent may vary, e.g., depending upon the desiredelastic properties and toughness, and in general ranges from about 0.01to about 30%, and in some embodiments from about 0.05 to about 20%, andin yet other embodiments to about 0.01 to about 6% (w/v).

The implant may then be further formed or shaped (with appropriatedimensions) for the intended clinical purpose, as known in the art. Insome embodiments, the pepsinized collagen membrane may be included in amulti-layer structure that contains a backing layer, for example, whichserves to shield the collagen membrane from the in-growth of nativetissue cells from one side. See, e.g., Shu-Tung Li, BiologicBiomaterials: Tissue-Derived Biomaterials (Collagen). In: BiomedicalEngineering Handbook, Ed. J. D. Bronzino, 42-1 to 42-23, CRC Press, Inc.Boca Raton, Fla., 2000. The implants may be sterilized by methods knownin the art, e.g., gamma irradiation, e-beam, ethylene oxide (EtO), steamand dry heat, and packaged (or vice versa). The packages may contain ina separate container (e.g., a vial) a therapeutically active orbeneficial agent so it can be added to (e.g., disposed on) the implantjust prior to implantation.

The resulting implants may be employed in a variety of clinicalapplications known in the art. See, e.g., U.S. Pat. No. 6,716,225 (nerverepair); U.S. Pat. No. 7,807,192 (repair of pericardium tissue afteropen-heart surgery, repair of hernia of the abdominal-wall); U.S. Pat.No. 7,374,777 (repairing meningeal tissue); U.S. Pat. No. 6,752,834(reconstruction of bone or cartilage tissue). The collagen sponges, forexample, may be useful in hemostasis and wound closure. The collagenmembranes may be useful in soft tissue repair constructions thatordinarily utilize artificial membranes, such as skin graftingprocedures (e.g., burned skin replacements), tendon reconstruction, andwound repair. They may also be shaped into various forms and used inconnection with hard tissue repair. The membranes may be used in theirtwo-dimensional configuration by successively packing the membranes intoa defect, such as a cranial or periodontal cavity. The membranes may bereformed into three dimensional objects for implantation in replacingsections of bone by rolling into cylinders, or by stacking and cuttingto shape.

Pepsinized collagen implants that have anti-adhesive properties may beparticularly advantageous in various clinical applications, e.g., as ananti-adhesion barrier for separating various tissue and organs, or as asurgical guide that is place in vivo at a site in which multiplesurgical procedures are to be performed over a period of time. Forinstance, in certain spinal procedures, the collagen membranes of thepresent invention may be folded and placed in vivo over the spine aftera procedure, such as a laminectomy, is performed, and which needs to bere-accessed. In this instance, the folded membrane is placed such thatit extends from a perpendicular plane of the surgical site up to thesurface of the skin. In the subsequent surgery at a later time, theedges of the membrane can be followed for direct access to the surgicalsite providing the surgeon with an unblocked path to the spine.

Yet other clinical applications that may exploit the properties of theanti-adhesive implants include gynecological procedures, abdominalsurgical procedures, inguinal or femoral hernia repair, and in thetreatment of large burns. For example, in the course of gynecologicaland abdominal surgical procedures, the membrane can be applied directlyto the organ that is operated upon, and which separates the organ fromthe surrounding tissue, as it slowly dissolves over the course of time,e.g., which in some embodiments is from about 2 to about 12 weeks,during which time the organ or tissue is allowed to heal withoutadhesion to the surrounding tissue.

The invention is now described in terms of the following non-limitingexamples. Example 1 describes the preparation of a buffer. Example 2describes preparation of a pepsinized collagen sponge. Examples 3-14describe preparation of pepsinized collagen membranes.

Example 1

Sodium acetate (4.1 g), calcium propionate (5.906 g) and sorbitol (50 g)were dissolved in 400 ml of deionized water in volumetric flask. Thevolume was adjusted to 500 ml mark by adding additional water. The pH ofthe solution was adjusted to 7.4 by 0.01 N HCl solution. This isreferred to hereinafter as the “sorbitol buffer.”

Example 2

Wet pepsinized collagen (10 g) was mixed with sorbitol buffer (40 ml) atpH 7.4. The viscous soluble solution was poured into a tray andlyophilized. The resultant sponge was cross-linked by EDCI in alcohol(100 ml) and triethylamine (100 microliters) at room temperature for 6hours. The sponge was washed with water and alcohol and dried at roomtemperature under vacuum. The resultant sponge was highly porous,flexible, and had a full or fluffy appearance.

Example 3

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. The viscous transparent solution was poured into a trayand air dried at room temperature. The resultant membranes werecross-linked by EDCI in alcohol (100 ml) and triethylamine (100microliters) at room temperature for 6 hours. The membranes were washedwith water and alcohol to give transparent membranes. This was dried atroom temperature under vacuum.

Example 4

Wet pepsinized collagen (10 g) was mixed with sorbitol buffer (2 ml) atpH 7.4. The viscous semi-soluble solution was poured into a tray andlyophilized. The resultant membrane was cross-linked by EDCI in alcohol(100 ml) and triethylamine (100 microliters) at room temperature for 6hours. The membrane was washed with water and alcohol to give asemi-transparent membrane. This was dried at room temperature undervacuum. The membrane was smooth and thin in appearance.

Example 5

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 800 mg of D-glucosamine HCl was added and mixedto dissolve it. The viscous transparent solution was poured into a trayand air dried at room temperature. The resultant membranes werecross-linked by EDCI in alcohol (100 ml) and triethylamine (100microliters) at room temperature for 6 hours. The membranes were washedwith water and alcohol to give transparent membranes havinganti-adhesive properties. This was dried at room temperature undervacuum.

Example 6

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 800 mg of D-glucosamine HCl was added and mixedto dissolve it. The viscous transparent solution was poured into a trayand air dried at room temperature. The resultant membranes werecross-linked by EDCI in alcohol (100 ml) and triethylamine (100microliters) at room temperature for 6 hours. The membranes were washedwith water and alcohol to give transparent membranes havinganti-adhesive properties. This was dried at room temperature undervacuum.

Example 7

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 600 mg of poly (ethyleneglycol) bis(carboxymethyl) ether, Mn 600, was added and mixed to dissolve it. Theviscous transparent solution was poured into a tray and air dried atroom temperature. The resultant membranes were cross-linked by EDCI inalcohol (100 ml) and triethylamine (100 microliters) at room temperaturefor 6 hours. The membranes were washed with water and alcohol to givetransparent membranes having anti-adhesive properties. This was dried atroom temperature under vacuum.

Example 8

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this 1 g of poly (ethyleneglycol) bis-(3-aminopropyl)terminated, Mn 1500, was added and mixed to dissolve it. The viscoustransparent solution was poured into a tray and air dried at roomtemperature. The resultant membranes were cross-linked by EDCI inalcohol (100 ml) and triethylamine (100 microliters) at room temperaturefor 6 hours. The membranes were washed with water and alcohol to givetransparent membranes having anti-adhesive properties. This was dried atroom temperature under vacuum.

Example 9

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 1 g of D-glucuronic acid was added and mixed todissolve it. The viscous transparent solution was poured into a tray andair dried at room temperature. The resultant membranes were cross-linkedby EDCI in alcohol (100 ml) and triethylamine (100 microliters) at roomtemperature for 6 hours. The membranes were washed with water andalcohol to give transparent membranes having anti-adhesive properties.This was dried at room temperature under vacuum.

Example 10

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 0.4 g of D-glucuronic acid and 0.4 g ofD-glucosamine were added and mixed to dissolve it. The viscoustransparent solution was poured into a tray and air dried at roomtemperature. The resultant membranes were cross-linked by EDCI inalcohol (100 ml) and triethylamine (100 microliters) at room temperaturefor 6 hours. The membranes were washed with water and alcohol to givetransparent membranes having anti-adhesive properties. This was dried atroom temperature under vacuum.

Example 11

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this 0.2 g of D-glucuronic acid, 0.2 g ofD-glucosamine and 0.1 g of L-lysine were added and mixed to dissolve it.The viscous transparent solution was poured into a tray and air dried atroom temperature. The resultant membranes were cross-linked by EDCI inalcohol (100 ml) and triethylamine (100 microliters) at room temperaturefor 6 hours. The membranes were washed with water and alcohol to givetransparent membranes having anti-adhesive properties. This was dried atroom temperature under vacuum.

Example 12

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 0.25 g of succinic anhydride was added followedby 200 microliters of triethylamine. The viscous transparent solutionwas poured into a tray and air dried at room temperature. The resultantmembranes were cross-linked by EDCI in alcohol (100 ml) andtriethylamine (100 microliters) at room temperature for 6 hours. Themembranes were washed with water and alcohol to give transparentmembranes having anti-adhesive properties. This was dried at roomtemperature under vacuum.

Example 13

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 50 mg of PEG-amine (8 arm, mol wt 20 K) wasadded and the mixture was vortexed to dissolve. The viscous transparentsolution was poured into a tray and air dried at room temperature. Theresultant membranes were cross-linked by EDCI in alcohol (100 ml) andtriethylamine (100 microliters) at room temperature for 6 hours. Themembranes were washed with water and alcohol to give transparentmembranes having anti-adhesive properties. This was dried at roomtemperature under vacuum.

Example 14

Wet pepsinized collagen (10 g) was solubilized in sorbitol buffer (40ml) at pH 7.4. To this, 150 mg of polyglutamic acid was added and themixture was vortexed to dissolve. The viscous transparent solution waspoured into a tray and air dried at room temperature. The resultantmembranes were cross-linked by EDCI in alcohol (100 ml) andtriethylamine (100 microliters) at room temperature for 6 hours. Themembranes were washed with water and alcohol to give transparentmembranes having anti-adhesive properties. This was dried at roomtemperature under vacuum.

All patent publications and non-patent publications are indicative ofthe level of skill of those skilled in the art to which this inventionpertains. All these publications are herein incorporated by reference tothe same extent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

The invention claimed is:
 1. A method of preparing a collagen membranefor use in tissue repair, comprising: (a) digesting collagen withpepsin, thus producing pepsinized collagen; (b) solubilizing thepepsinized collagen in a buffer composition comprising a polyol, whereinthe buffer has a substantially neutral pH and wherein the collagen ispresent in the buffer composition in an amount of about 2 mg to about 20mg per ml of the buffer; (c) drying the thus solubilized, pepsinizedcollagen; and (d) reacting the collagen of (c) with a cross-linkingagent, thus producing the collagen membrane, wherein the cross-linkingagent is added to the solubilized, pepsinized collagen in aconcentration of about 0.01% to about 6% (w/v).
 2. A pepsinized collagenmembrane, made by the method of claim 1, wherein the membrane comprisesan anti-adhesive agent.
 3. The membrane of claim 2, wherein the membraneseparates tissues and organs when placed in vivo.
 4. A method forpromoting tissue regeneration, for promoting tissue repair, or forpromoting or facilitating a tissue graft, comprising covering an area ofdamaged, injured, diseased wounded, removed or missing tissue of a bodyof a subject, with the pepsinized collagen membrane of claim 2.